A class of aromatic guanylhydrazones has been described in U.S. Pat. No. 5,750,573, the disclosure of which is incorporated by reference herein. These compounds have been shown to have therapeutic activity, primarily as anti-inflammatory agents. The activity is based upon an ability to suppress proinflammatory cytokine synthesis in activated macrophages and other cells. The preferred compound, called "CNI-1493" (N,N'-bis[3,5-bis[1-(aminoiminomethyl)hydrazonoethyl]phenyl]decanediamide tetrahydrochloride), inhibits TNF (tumor necrosis factor) translation and suppresses the production of IL-1, IL-6, MIP-1.alpha. and MIP-1.beta. (proinflammatory cytokines) in human peripheral blood mononuclear cells. In vivo, CNI-1493 protects mice against lethal effects of endotoxin (lipopolysaccharide, LPS) and is anti-inflammatory as evidenced by suppression of rat paw edema and inflammation following carageenan administration. Therefore, there is always a need to improve the therapeutic properties of drug candidates such as CNI-1493 and related aromatic guanylhydrazone compounds.
Macrophages play important roles in fundamental immune protection, including, for example, phagocytosis, antigen presentation, microbiocidal and tumoricidal activities, and release of a large number of factors during host defense and inflammation. For example, upon stimulation by interferon .gamma. (INF-.gamma.) and potent macrophage activators such as bacterial endotoxin, macrophages secrete large amounts of reactive nitrogen intermediates (RNI), and several cytokines including TNF-.alpha., IL-1, IL-6, MIP-1.alpha., MIP-1.beta. that augment the inflammatory response during bacterial infection. Generation of RNI and tumor cell killing by macrophages are both dependent on L-arginine (Keller et al., Cancer Res. 50:1421-1425, 1990; Hibbs et al., J. Immunol. 140:550-565, 1987; Drapier et al., J. Immunol. 140:2829-2838, 1988), a common substrate shared by arginase and nitric oxide synthase (NOS). Arginase (EC 3.5.3.1.) is an enzyme active in converting L-arginine to L-ornithine and urea, and thus plays an essential role in the urea cycle, as well as the biosynthesis of proline and polyamines (Janne et al., Ann. Med. 23:241, 1991). Nitric oxide synthase is an enzyme active in catalyzing the formation of nitric oxide (NO). Nitric oxide synthase is an important molecule implicated in antimicrobial, cytotoxic, and inflammatory processes mediated by macrophages, as well as in blood pressure regulation and neurotransmission in the nervous system (Hibbs et al., Biochem. Biophys. Res. Comm. 157:87-94, 1988; Moncada et al., Proc. Natl. Acad. Sci. USA 88:2166-2170, 1991, Nathan, FASEB J. 6:3015-3064, 1992; Moncada et al., N. Engl. J. Med. 329:2001-2012, 1993). Both arginase (AII) and NO synthase (iNOS) are LPS-inducible in macrophages (Granger et al., J. Clin. Invest. 85:264-273, 1990; Currie, Nature 273:758-759, 1978; Corraliza, J. Immunol. Methods 174:231-235, 1994; Stuehr et al., Proc. Natl. Acad. Sci. USA 82:7738-7742, 1985; Steuhr et al., J. Immunol. 139:518-525, 1987; Modlell, Immunology Lett. 40:139-146, 1994). Although a biological role for urea and NO.sub.2.sup.- /NO.sub.3.sup.- synthesis by cells of the immune system is not yet known, it appears to represent a quantitative feature in the activation of macrophages. Arginase activity potentially controls the NO synthesis of macrophages by substrate depletion as in the absence of arginine the production of NO is dramatically reduced (Vodovotz, J. Immunol. 152:4110-4118, 1994). Thus, all factors controlling the relative rates of flux of L-arginine between arginase and NOS may be important in the regulation of macrophage's cytotoxic activity.
L-arginine transport in macrophages and other cells also increases in response to agents that induce iNOS (Bogel, Biochem J. 284:15-18, 1992; Sato et al., Biochem. Biophys. Acta 1069:46-52, 1991; and Cendan, Surgery 117:213-219, 1995). CNI-1493 and related compounds inhibit cytokine-induced arginine transport (IC.sub.50 =59 .mu.M), and NO production (IC.sub.50 =5 .mu.M. The cytokine-suppressive effects of CNI-1493 are possibly not mediated by inhibition of NO or L-arginine transport, nor by generalized suppression of protein synthesis or RNA synthesis.
Fetuin is a globular protein of about 60-70 kDa, containing 20-25% carbohydrate (by weight) and characterized by multiple internal disulfide bonds. The human fetuin sequence (also known as .alpha.2-HS glycoprotein) is provided herein as SEQ ID NO. 1 and SEQ ID NO. 2. Fetuin was first identified over 50 years ago as a major protein component of fetal bovine serum but its biological function remains unclear. Bovine fetuin is a globular 341-amino acid polypeptide with six internal disulfide bonds and three N-linked and two O-linked oligosaccharide chains. Primary amino acid sequence and the position of cysteine residues are well conserved in human, bovine, sheep, rat and mouse fetuin homologs (Dziegielewska et al., J. Biol. Chem. 265:4354, 1990; Rauth et al., Eur. J. Biochem. 205:321, 1992; Lee et al., Proc. Natl. Acad. Sci. USA 84:4403, 1987; and Brown et al., Eur. J. Biochem. 205:321, 1992). Fetuin levels in human plasma are regulated in a manner of a negative acute phase reactant (Lebreton et al., J. Clin. Invest. 64:1118, 1979). IL-1 was shown to suppress fetuin transcript levels in cultured hepatocytes (Akhoundi et al., J. Biol. Chem. 268:15925, 1994). Fetuin appears to be expressed in bone because transcripts have been detected in both chondrocytes and osteoblasts (Yang et al., Bone 12:7, 1991). The polypeptide .alpha.2-HS glycoprotein is a human homolog of fetuin and is secreted in high levels by adult liver into the peripheral circulation (Triffitt et al., Nature 262:226, 1976).
Human fetuin has 3 N-linked oligosaccharide chains (attached to the amine nitrogen atom of asparagine), and 2 O-linked oligosaccharide chains (attached to the oxygen atom of serine or threonine). The sugar moiety directly attached to the fetuin polypeptide is usually a N-acetylglucosamine residue. The terminal sugar residue is usually a sialic acid, in particular a N-acetylneuraminic acid (NANA) residue, which bears a net negative charge. If one removes the terminal sialic acid residue from fetuin by neuraminidase treatment, the resulting glycoprotein is an asialofetuin. Fetuin is a carrier protein for growth factors, and human fetuin is sometimes referred to as .alpha.2-HS-glycoprotein. Thus, it is considered that fetuin's biological effects on cultured cells are related to its carrier function for molecules with growth-promoting properties. Fetuin is secreted by adult liver into the peripheral circulation and accumulates to high levels in bone.
The synthesis of human .alpha.2-HS-glycoprotein is down-regulated by cytokines (hIL-1.beta., hIL-6) (Lebreton et al., J. Clin. Invest. 64:1118-1129, 1979). Human fetuin levels are decreased (25-50%) in trauma patients (van Oss et al., J. Trauma 15:451, 1975). Therefore, there is a need in the art to find a utility for fetuin and to understand fetuin's physiological role and the importance of its many negatively charged (at physiologic pH) sialic acid residues.